US5527688A - Rapid assays for protein kinase activity - Google Patents

Rapid assays for protein kinase activity Download PDF

Info

Publication number
US5527688A
US5527688A US08/225,467 US22546794A US5527688A US 5527688 A US5527688 A US 5527688A US 22546794 A US22546794 A US 22546794A US 5527688 A US5527688 A US 5527688A
Authority
US
United States
Prior art keywords
peptide
enzyme
solid phase
protein kinase
phosphorylated
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
Application number
US08/225,467
Other languages
English (en)
Inventor
A. Krishna Mallia
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Pierce Biotechnology Inc
Original Assignee
Pierce Chemical Co
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Pierce Chemical Co filed Critical Pierce Chemical Co
Priority to US08/225,467 priority Critical patent/US5527688A/en
Priority to AU22346/95A priority patent/AU2234695A/en
Priority to PCT/US1995/003988 priority patent/WO1995027796A1/fr
Assigned to PIERCE CHEMICAL COMPANY reassignment PIERCE CHEMICAL COMPANY ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: MALLIA, KRISHNA
Application granted granted Critical
Publication of US5527688A publication Critical patent/US5527688A/en
Assigned to PIERCE BIOTECHNOLOGY, INC. reassignment PIERCE BIOTECHNOLOGY, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: PIERCE CHEMICAL COMPANY
Assigned to PIERCE BIOTECHNOLOGY, INC. reassignment PIERCE BIOTECHNOLOGY, INC. CHANGE OF NAME (SEE DOCUMENT FOR DETAILS). Assignors: PIERCE CHEMICAL COMPANY
Assigned to DEUTSCHE BANK AG, NEW YORK BRANCH reassignment DEUTSCHE BANK AG, NEW YORK BRANCH SECURITY INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: PIERCE BIOTECHNOLOGY, INC.
Assigned to HYCLONE LABORATORIES, INC., PIERCE BIOTECHNOLOGY, INC. reassignment HYCLONE LABORATORIES, INC. RELEASE OF SECURITY INTEREST Assignors: DEUTSCHE BANK AG NEW YORK BRANCH
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

Links

Images

Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5021—Test tubes specially adapted for centrifugation purposes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase

Definitions

  • the present invention relates to techniques for assaying enzymatic activity and, more particularly, to rapid radioactive or non-radioactive methods for measuring the activity of protein kinases.
  • Protein kinases are enzymes which covalently modify proteins and peptides by the attachment of a phosphate group to one or more sites on the protein or peptide. The measurement of protein kinase activity is important since studies have shown that these enzymes are key regulators of many cell functions.
  • the most widely used technique for measuring protein kinase activity is based on radioactive detection.
  • a sample containing the kinase of interest is incubated with activators and a substrate in the presence of gamma 32 P-ATP. After a suitable incubation period, the reaction is stopped and an aliquot of the reaction mixture is placed directly onto a filter which binds the substrate. The filter is then washed multiple times to remove excess radioactivity, and the amount of radiolabelled phosphate incorporated into the substrate is measured by scintillation counting.
  • This method is widely used and provides an accurate method for determining protein kinase activity in both crude and purified samples.
  • multiple washings which are generally done by manually transferring the filter to a beaker and washing and rinsing with gentle agitation, the procedure is quite time consuming.
  • the present invention provides an improvement in a method of measuring enzymatic activity of a Protein Kinase, such as Protein Kinase A, Protein Kinase C, and tyrosine kinases.
  • the general method to which the improvement of the present invention is directed comprises (1) phosphorylating a peptide substrate in an aqueous medium in the presence of a phosphoryl donor compound and the enzyme, (2) while in said aqueous medium, adsorbing the phosphorylated peptide to a solid phase, (3) washing the solid phase with a wash solution to remove non-adsorbed constituents which would interfere with the measurement of enzyme activity and (4) measuring the amount of phosphorylated peptide adsorbed to the solid phase.
  • the improvement to the foregoing method provided by this invention involves, first, using as the solid phase a membrane positioned within a chamber which separates the chamber into discrete first and second regions and, second, accomplishing the washing step by passing, with applied external force, the wash solution through the membrane from the first region to the second region.
  • the improvement contributed by this invention increases the speed with which the assay can be accomplished, and is applicable with respect to radioactive assays and assays based on charge separation.
  • a new non-radioactive method for measuring enzymatic activity of Protein Kinases involves forming an aqueous solution of a peptide substrate, a phosphoryl donor compound, and the enzyme.
  • the peptide can be phosphorylated in the presence of the donor and enzyme, and has previously been chemically modified to contain a dye which permits spectrophotometric or fluorometric detection.
  • the solution is incubated for a sufficient time and under conditions whereby the peptide is phosphorylated through action of the enzyme. Thereafter, the phosphorylated peptide is separated from solution by adsorbing the peptide on a solid phase having the Fe +3 ion chelated to the phase. Subsequently, the solid phase is washed to remove non-phosphorylated peptide and the amount of phosphorylated peptide is measured spectrophotometrically or fluorometrically.
  • FIG. 1 illustrates the component parts of a unit which can be used in accomplishing the assays described herein;
  • FIG. 2 illustrates, in assembled form, the unit referred to above
  • FIGS. 3 and 4 are standard curves prepared from the assay described in Example VI.
  • FIGS. 1 and 2 depict a unit which can be used in accomplishing the assays described herein.
  • the unit is useful in those aspects of the assays described herein wherein phosphorylated peptide substrate is separated from other constituents by adsorption to a solid phase with subsequent washing to remove non-adsorbed elements.
  • the depicted unit 10 includes a tube 12 generally of a shape such that it can be accommodated in the receptacles of conventional centrifuges.
  • a cap 14 is provided to enclose the tube when desired.
  • a bucket 18 is provided for the purpose of housing a membrane 16 within the tube 12.
  • the membrane 16 rests flatly on the bottom surface of the bucket 18 and is held in place by the sleeve 20. While not specifically illustrated, the bottom surface of the bucket 20 is perforated to permit the passage of wash solution through the membrane and bottom surface.
  • the membrane 16 separates the chamber within the tube 12 into two discrete regions; a first region 22 being the interior of the bucket 18 and the second region 24 being the volume of the chamber not occupied by the bucket.
  • wash solution placed in the first region 22 is forced through the membrane 16 and into the second region 24.
  • the force to accomplish this is preferably applied centrifugally by use of a centrifuge.
  • the assay is radioactive
  • phosphocellulose paper such as P81 from Whatman
  • the assay is accomplished conventionally except with respect to washing free radioactive donor compound, typically 32 P-ATP, from the membrane. As indicated, this is accomplished by forcing the wash solution, using applied force, preferably centrifigual, through the membrane. The result is that the washing time necessary to achieve acceptable background levels is reduced, as is handling of the radioactive membrane. In this latter aspect, after washing, the bucket can be directly transferred to a scintillation vial without membrane removal.
  • a non-radioactive method for assaying Protein Kinase activity is a non-radioactive method for assaying Protein Kinase activity.
  • a peptide substrate is phosphorylated in aqueous solution by a Protein Kinase through incubation with the enzyme and a phosphoryl donor compound.
  • Non-radioactive ATP is most commonly used as the donor.
  • the former is adsorbed to a solid phase containing the Fe +3 ion which has specific affinity for the phosphoryl group.
  • the Fe +3 iron is chelated to the solid phase through iminodiacetic acid groups which are covalently attached to the phase.
  • the phase is typically customary filter paper. After adsorption, the phase is washed to remove non-phosphorylated peptide.
  • the peptide substrate prior to phosphorylation is chemically modified with a dye so that the amount of phosphorylated derivative adsorbed to the solid phase can, after washing to remove the non-phosphorylated constituent, be spectrophotometrically or fluorometrically measured.
  • a dye so that the amount of phosphorylated derivative adsorbed to the solid phase can, after washing to remove the non-phosphorylated constituent, be spectrophotometrically or fluorometrically measured.
  • Such measurement generally is made after the peptide has been removed, e.g., eluted, from the solid phase.
  • the non-phosphorylated constituent can be measured and the amount of peptide adsorbed can be deduced by difference.
  • an essential aspect of the non-radioactive assay disclosed herein resides in the ability to measure activity spectrophotometrically or fluorometrically. As opposed to other methods, such as gel electrophoresis or HPCL, this method is less cumbersome and uses inexpensive equipment readily available in most laboratories.
  • the dye used to modify the peptide must not only be detectable by the identified means, but it must not interfere with phosphorylated peptide adsorption to the Fe +3 modified support.
  • a useful dye is Lissamine Rhodamine B sulfonyl chloride.
  • Examples I & II illustrate the invention with respect to an assay using radioactive detection.
  • the remaining examples illustrate use of the invention with a non-radioactive protocol.
  • membranes were prepared by cutting small circles (8 mm diameter) out of phosphocellulose paper obtained from Whatman under their designation P81 paper. Using the tube and bucket arrangement depicted in the drawings, the membranes were inserted into the illustrated buckets thus covering the perforated bottom surface of the buckets. Thereafter, insertion of the sleeve holds the membranes in place.
  • Lipid Buffer (8 mole % L-alpha Phosphatidyl-L-serine and 24 ⁇ g/ml phorbol 12-myristate 13-acetate in 50 mM Tris, pH 7.5)
  • Peptide Buffer (900 ⁇ M peptide-RKRTLRRL, EGF receptor--in 50 mM Tris buffer, pH 7.5)
  • ATP Buffer 150 ⁇ M ATP and 45 mM magnesium acetate in 50 mM Tris buffer
  • Radioactive gamma- 32 P-ATP ( 32 P-ATP) was also obtained from Amersham and used in accordance with the instructions for the assay (4 ⁇ l of 32 P-ATP was added to 500 ⁇ l of the ATP buffer contained in the kit).
  • reaction solution was then formed by combining 25 ⁇ l of the mixture with 25 ⁇ l of 32 P-ATP solution and 25 ⁇ l of Protein Kinase C sample. The reaction proceeded for 15 minutes at room temperature and was then terminated by the addition of 100 ⁇ l 75 mM phosphoric acid.
  • a magnesium chloride-ATP buffer (see example VI) was rendered radioactive by the addition of 1 ⁇ l of 32 P-ATP per 100 ⁇ l of buffer. 5 ⁇ l of this radioactive buffer, 5 ⁇ l of an activator solution (500 ⁇ M c-AMP in water) and 5 ⁇ l of Kemptide solution (1 mg/ml Kemptide-LRRASLG, amino acid sequence) were added to 10 ⁇ l Protein Kinase A sample. After 15 minutes incubation, the reaction was quenched by addition of 35 ⁇ l 75mM phosphoric acid.
  • Example I protocol Preparation of a standard curve as described with respect to Example I indicates that the assay is linear with a regression coefficient of 0.994.
  • Toomik et. al.* was utilized as follows:
  • Whatman 3MM filter paper cut in circles of 30 cm in diameter, is activated by first immersing in NaOH for two minutes. For twenty five circles, 7500 ml of NaOH (3M) are used. Following the initial immersion, 3000 ml of epichlorohydrin is added and the mixture incubated with gentle agitation for two hours.
  • the circles are washed with one liter of deionized water each.
  • the circles of paper are then added to 7500 ml of a 0.7M sodium carbonate solution containing 360 g of iminodiacetic acid.
  • the paper is gently agitated for two hours, then allowed to incubate overnight with no agitation.
  • the circles are again washed with one liter of deionized water each, and then added to 8500 ml of a 50 mM ferric chloride hexahydrate solution. The circles are incubated with gentle agitation for one hour.
  • the circles are then washed sequentially with one liter of 1M NaCl each followed by one liter of deionized water each.
  • the circles are allowed to air dry in a dark room and then isolated in an opaque, sealed container.
  • the iminodiacetic acid/ferric iron membranes prepared as in Example I, are placed in the bottom of a small bucket which has a perforated bottom.
  • the membranes are held in place with the use of sleeves which are inserted into the bucket.
  • the buckets are placed into microcentrifuge tubes.
  • Kemptide a well known peptide substrate for Protein Kinase A (PKA), was conventionally synthesized by the solid phase approach using Fmoc protected amino acids. Following synthesis, the peptide was derivatized with Lissamine Rhodamine B sulfonyl chloride as follows:
  • NMP N-methylpyrrolidone
  • a fresh solution of Lissamine Rhodamine B sulfonyl chloride dye is prepared in NMP by dissolving two equivalents of the dye in ⁇ 1 ml of NMP.
  • An activator solution consisting of a mixture of NMP (0.025 ml), triethylamine (0.025 ml) and dimethylaminopyridine (2 mg) is added to the fresh solution to form an activated dye solution.
  • the activated dye solution is then added to the resin and reacted at room temperature for 18 hours. Following the reaction, the resin is washed with dimethylformamide until the wash is colorless. The resin is then washed three times with dichloromethane and dried under vacuum. The peptide is then cleaved from the resin using standard procedures.
  • a reaction mixture was prepared by mixing 10 ⁇ l of Reaction Buffer (10 mM ATP, 50 mM MgCl 2 , 0.01% Triton X-100, and 100 mM Tris, pH 7.4), 10 ⁇ l Activator solution (500 ⁇ M cyclic-AMP in water), and 10 ⁇ l dye modified Kemptide per Example 3 (in solution as 550 ⁇ g peptide in 550 ⁇ l of deionized water).
  • 20 ⁇ l of Protein Kinase A containing sample is added to the mixture followed by incubation at 30° C. for 30 minutes and then boiling for five minutes to terminate the reaction. Thereafter, 20 ⁇ l of the mixture containing phosphorylated peptide is applied to the membrane contained in the bucket within the microcentrifuge tubes per Example IV. Alternatively, instead of boiling, the sample may be applied to the membrane immediately, effectively stopping the reaction.
  • phosphopeptide binding buffer (0.1M sodium acetate, 0.5M sodium chloride, 0.02% sodium azide, pH5.0) is applied to the membrane and allowed to incubate for three minutes.
  • the tube containing the bucket is then placed in a conventional centrifuge and the binding buffer washed through the membrane by centrifugation at 6500 rpm for one minute. This wash step is then repeated for a second time.
  • the bucket is transferred to a clean receptacle and the phosphorylated peptide is eluted from the membrane using 250 ⁇ l of an elution buffer (0.1M ammonium bicarbonate and 0.02% sodium azide, pH 8.0).
  • the elution buffer is incubated and washed through the membrane in the same manner as the binding buffer.
  • the elution step is then repeated for a second time.
  • Conventional spectrophometric measurement of absorbance of the eluate can be made at 570 nm.
  • a fluorescent measurement can be employed using excitation at 573 nm and emission at 589 nm (5 nm window).

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Zoology (AREA)
  • Clinical Laboratory Science (AREA)
  • Hematology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
US08/225,467 1994-04-08 1994-04-08 Rapid assays for protein kinase activity Expired - Lifetime US5527688A (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
US08/225,467 US5527688A (en) 1994-04-08 1994-04-08 Rapid assays for protein kinase activity
AU22346/95A AU2234695A (en) 1994-04-08 1995-03-31 Rapid assays for protein kinase activity
PCT/US1995/003988 WO1995027796A1 (fr) 1994-04-08 1995-03-31 Evaluation rapide de l'activite de la proteine kinase

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
US08/225,467 US5527688A (en) 1994-04-08 1994-04-08 Rapid assays for protein kinase activity

Publications (1)

Publication Number Publication Date
US5527688A true US5527688A (en) 1996-06-18

Family

ID=22844984

Family Applications (1)

Application Number Title Priority Date Filing Date
US08/225,467 Expired - Lifetime US5527688A (en) 1994-04-08 1994-04-08 Rapid assays for protein kinase activity

Country Status (3)

Country Link
US (1) US5527688A (fr)
AU (1) AU2234695A (fr)
WO (1) WO1995027796A1 (fr)

Cited By (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5763198A (en) * 1994-07-22 1998-06-09 Sugen, Inc. Screening assays for compounds
US5869275A (en) * 1998-07-20 1999-02-09 Huang; Eric Z. Affinity ultrafiltration assay for transferase activity
US6280965B1 (en) * 1999-05-26 2001-08-28 Nova Southeastern University Simple non-radioactive assay for estimating protein kinase C and protein phosphatase-1
US6297018B1 (en) 1998-04-17 2001-10-02 Ljl Biosystems, Inc. Methods and apparatus for detecting nucleic acid polymorphisms
US6410255B1 (en) 1999-05-05 2002-06-25 Aurora Biosciences Corporation Optical probes and assays
WO2003020747A1 (fr) * 2001-09-06 2003-03-13 Cnrs (Centre National De La Recherche Scientifique) Peptides modifies et leur utilisation dans le traitement de maladies auto-immunes
US20030100029A1 (en) * 2001-11-09 2003-05-29 Clinical Reference Laboratory Methods of determining active levels of drugs in fluid samples
US20030175815A1 (en) * 1999-05-21 2003-09-18 Caliper Technologies Corp. Assay methods and systems
US6689565B2 (en) 1999-05-21 2004-02-10 Caliper Technologies Corp. Assay methods and systems
US20040038306A1 (en) * 2002-05-03 2004-02-26 Brian Agnew Compositions and methods for detection and isolation of phosphorylated molecules
US6720162B2 (en) 2000-05-31 2004-04-13 Promega Corporation Assay for kinases and phosphatases
US20050009124A1 (en) * 2001-11-26 2005-01-13 Echelon Biosciences Incorporated Assays for detection of phosphoinositide kinase and phosphatase activity
US20050064485A1 (en) * 2003-09-12 2005-03-24 Kurt Vogel Multiplex binding and activity assays
US20070054304A1 (en) * 2002-05-03 2007-03-08 Invitrogen Corporation Compositions and methods for detection and isolation of phosphorylated molecules
US20070154980A1 (en) * 2005-12-30 2007-07-05 Gasper Susan M Fluorescent dyes
US20080233555A1 (en) * 2002-06-14 2008-09-25 Thermo Fisher Scientific (Rockford) Llc Homogeneous assay for enzymatic activity
US7582461B2 (en) 2003-07-29 2009-09-01 Life Technologies Corporation Kinase and phosphatase assays
US7619059B2 (en) 2003-07-29 2009-11-17 Life Technologies Corporation Bimolecular optical probes
US7727752B2 (en) 2003-07-29 2010-06-01 Life Technologies Corporation Kinase and phosphatase assays
US7745142B2 (en) 1997-09-15 2010-06-29 Molecular Devices Corporation Molecular modification assays
CN105921197A (zh) * 2016-06-21 2016-09-07 长江大学 一种用于相分离的多刻度离心管
CN106999615A (zh) * 2014-11-28 2017-08-01 通用电气医疗集团股份有限公司 包含金属络合物Gd‑DOTA的葡甲胺盐的制剂
US10213482B2 (en) 2014-12-12 2019-02-26 Immupharma France Sa Methods of treating chronic inflammatory diseases

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE59810846D1 (de) * 1997-03-21 2004-04-01 Biotechnolog Forschung Gmbh Verbindungen zum nachweis von phosphorsäureestern
US8778614B2 (en) 2010-08-24 2014-07-15 Enzo Life Sciences, Inc. Assays for detecting modified compounds

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5120644A (en) * 1986-04-01 1992-06-09 Wako Pure Chemical Industries, Ltd. Peptide derivatives and activity measuring method of physiologically active substances using the same as substrates
WO1993010461A1 (fr) * 1991-11-12 1993-05-27 Promega Corporation Determination non radioactive d'enzymes

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4923802A (en) * 1985-06-13 1990-05-08 Immunex Corporation Peptide substrates for the detection, characterization and purification of protein kinase C
US5141852A (en) * 1988-12-12 1992-08-25 The United States Of America As Represented By The Department Of Health And Human Services Assay of protein kinases with peptide substrates
IL88831A0 (en) * 1988-12-29 1989-07-31 Yeda Res & Dev Blood assay

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5120644A (en) * 1986-04-01 1992-06-09 Wako Pure Chemical Industries, Ltd. Peptide derivatives and activity measuring method of physiologically active substances using the same as substrates
WO1993010461A1 (fr) * 1991-11-12 1993-05-27 Promega Corporation Determination non radioactive d'enzymes

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
Casnellie, Methods in Enzymology, vol. 200, pp. 115 120, 1991. *
Casnellie, Methods in Enzymology, vol. 200, pp. 115-120, 1991.
Sigma catalog, Biochemicals and Organic Compounds for Research and Diagnostic Reagerts, pp. 1072 1073 and pp. 854 857, 1993. *
Sigma catalog, Biochemicals and Organic Compounds for Research and Diagnostic Reagerts, pp. 1072-1073 and pp. 854-857, 1993.
Toomik et al, Analytical Biochemistry, vol. 209, pp. 348 353, 1993. *
Toomik et al, Analytical Biochemistry, vol. 209, pp. 348-353, 1993.
Yano et al, Biochemical and Biophysical Research Communications, vol. 175, No. 3, Mar. 29, 1991, pp. 1144 1151. *
Yano et al, Biochemical and Biophysical Research Communications, vol. 175, No. 3, Mar. 29, 1991, pp. 1144-1151.

Cited By (44)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5763198A (en) * 1994-07-22 1998-06-09 Sugen, Inc. Screening assays for compounds
US7745142B2 (en) 1997-09-15 2010-06-29 Molecular Devices Corporation Molecular modification assays
US6297018B1 (en) 1998-04-17 2001-10-02 Ljl Biosystems, Inc. Methods and apparatus for detecting nucleic acid polymorphisms
US5869275A (en) * 1998-07-20 1999-02-09 Huang; Eric Z. Affinity ultrafiltration assay for transferase activity
US6410255B1 (en) 1999-05-05 2002-06-25 Aurora Biosciences Corporation Optical probes and assays
US20080146460A1 (en) * 1999-05-05 2008-06-19 Invitrogen Corporation Optical probes and assays
US20030087328A1 (en) * 1999-05-05 2003-05-08 Pollok Brian A. Optical probes and assays
US20030175815A1 (en) * 1999-05-21 2003-09-18 Caliper Technologies Corp. Assay methods and systems
US7150966B2 (en) 1999-05-21 2006-12-19 Caliper Life Sciences, Inc. Assay methods and systems
US7122659B2 (en) 1999-05-21 2006-10-17 Caliper Life Sciences, Inc. Assay methods and systems
US6689565B2 (en) 1999-05-21 2004-02-10 Caliper Technologies Corp. Assay methods and systems
US20040033531A1 (en) * 1999-05-21 2004-02-19 Caliper Technologies Corp. Assay methods and systems
US20070161071A1 (en) * 1999-05-21 2007-07-12 Caliper Life Sciences, Inc. Assay methods and systems
US6699655B2 (en) 1999-05-21 2004-03-02 Caliper Technologies Corp. Fluorescent polarization assays involving multivalent metal ions and systems
US20040058406A1 (en) * 1999-05-21 2004-03-25 Caliper Technologies Corp. Assay methods and systems
US6280965B1 (en) * 1999-05-26 2001-08-28 Nova Southeastern University Simple non-radioactive assay for estimating protein kinase C and protein phosphatase-1
US6720162B2 (en) 2000-05-31 2004-04-13 Promega Corporation Assay for kinases and phosphatases
US20040086954A1 (en) * 2000-05-31 2004-05-06 Said Goueli Assay for kinases and phosphatases
US6893834B2 (en) 2000-05-31 2005-05-17 Promega Corporation Assay for kinases and phosphatases
EP2143730A3 (fr) * 2001-09-06 2011-06-29 Centre National de la Recherche Scientifique (CNRS) Peptides modifiés et leur utilisation pour le traitement des maladies auto-immunes
US20030186849A1 (en) * 2001-09-06 2003-10-02 Zimmer Robert H. Modified peptides and their use for the treatment of autoimmune diseases
EP2143730A2 (fr) 2001-09-06 2010-01-13 Centre National De La Recherche Scientifique (Cnrs) Peptides modifiés et leur utilisation pour le traitement des maladies auto-immunes
WO2003020747A1 (fr) * 2001-09-06 2003-03-13 Cnrs (Centre National De La Recherche Scientifique) Peptides modifies et leur utilisation dans le traitement de maladies auto-immunes
US7094558B2 (en) 2001-11-09 2006-08-22 Clinical Reference Laboratory Methods of determining active levels of drugs in fluid samples
US20040146852A1 (en) * 2001-11-09 2004-07-29 Stout Robert L. Methods of determining active levels of drugs in fluid samples
US20030100029A1 (en) * 2001-11-09 2003-05-29 Clinical Reference Laboratory Methods of determining active levels of drugs in fluid samples
US20050009124A1 (en) * 2001-11-26 2005-01-13 Echelon Biosciences Incorporated Assays for detection of phosphoinositide kinase and phosphatase activity
US7102005B2 (en) 2002-05-03 2006-09-05 Molecular Probes, Inc. Compositions and methods for detection and isolation of phosphorylated molecules
US20040038306A1 (en) * 2002-05-03 2004-02-26 Brian Agnew Compositions and methods for detection and isolation of phosphorylated molecules
US20070054304A1 (en) * 2002-05-03 2007-03-08 Invitrogen Corporation Compositions and methods for detection and isolation of phosphorylated molecules
US7776533B2 (en) 2002-05-03 2010-08-17 Life Technologies Corporation Compositions and methods for detection and isolation of phosphorylated molecules
US20080233555A1 (en) * 2002-06-14 2008-09-25 Thermo Fisher Scientific (Rockford) Llc Homogeneous assay for enzymatic activity
US7582461B2 (en) 2003-07-29 2009-09-01 Life Technologies Corporation Kinase and phosphatase assays
US7619059B2 (en) 2003-07-29 2009-11-17 Life Technologies Corporation Bimolecular optical probes
US7727752B2 (en) 2003-07-29 2010-06-01 Life Technologies Corporation Kinase and phosphatase assays
US8067536B2 (en) 2003-07-29 2011-11-29 Life Technologies Corporation Kinase and phosphatase assays
US20050064485A1 (en) * 2003-09-12 2005-03-24 Kurt Vogel Multiplex binding and activity assays
US7781187B2 (en) 2005-12-30 2010-08-24 Corning Incorporated Fluorescent dyes
US20070154980A1 (en) * 2005-12-30 2007-07-05 Gasper Susan M Fluorescent dyes
CN106999615A (zh) * 2014-11-28 2017-08-01 通用电气医疗集团股份有限公司 包含金属络合物Gd‑DOTA的葡甲胺盐的制剂
US10314927B2 (en) * 2014-11-28 2019-06-11 Ge Healthcare As Formulations of metal complexes
CN106999615B (zh) * 2014-11-28 2020-11-17 通用电气医疗集团股份有限公司 包含金属络合物Gd-DOTA的葡甲胺盐的制剂
US10213482B2 (en) 2014-12-12 2019-02-26 Immupharma France Sa Methods of treating chronic inflammatory diseases
CN105921197A (zh) * 2016-06-21 2016-09-07 长江大学 一种用于相分离的多刻度离心管

Also Published As

Publication number Publication date
AU2234695A (en) 1995-10-30
WO1995027796A1 (fr) 1995-10-19

Similar Documents

Publication Publication Date Title
US5527688A (en) Rapid assays for protein kinase activity
Witt et al. Rapid protein kinase assay using phosphocellulose-paper absorption
Van den Bosch et al. A review on methods of phospholipase A determination
Corbin et al. Studies of two different intrachain cGMP-binding sites of cGMP-dependent protein kinase.
Sheffield et al. A solid-phase method for the quantitation of protein in the presence of sodium dodecyl sulfate and other interfering substances
US5856082A (en) Devices and methods for characterizing proteins and peptides
Kaushal et al. Effect of zwitterionic buffers on measurement of small masses of protein with bicinchoninic acid
EP0375454B1 (fr) Immunoessai enzymatique pour la mesure du niveau de cyclosporine A dans des échantillons de sang entier
Van Etten et al. Phosphohistidine as a stoichiometric intermediate in reactions catalyzed by isoenzymes of wheat germ acid phosphatase
US5141852A (en) Assay of protein kinases with peptide substrates
US4366242A (en) Method and agent for the immunological determination of enzymes
Finlay et al. Kinetic and structural studies of hydroxyproline 2-epimerase
US5538858A (en) Rapid assay for radioactive determination of protein kinase activity
Morin Improved separation of creatine kinase cardiac isoenzyme in serum by batch fractionation.
Lam et al. Sequence homology analysis of a heterogenous protein population by chemical and enzymic digestion using a two-dimensional sodium dodecyl sulfate-polyacrylamide gel system
Hart Jr et al. Isolation of a protein component of sodium-potassium transport adenosine triphosphatase containing ligand-protected sulfhydryl groups
US4049496A (en) Rapid separation of plasma creatine kinase isoenzymes
US4808522A (en) Enzyme immunoassays using inorganic pyrophosphatase
JPH0215827B2 (fr)
Jergil et al. Biospecific affinity chromatography of an adenosine 3′: 5′-cyclic monophosphate-stimulated protein kinase (protamine kinase from trout testis) by using immobilized adenine nucleotides
US5693291A (en) Reagents kit for the quantitative analysis of proteins or/and peptides
Suh et al. Fluorescence studies of the binding of alkyl and aryl phosphates to rat muscle aldolase
SE454597B (sv) Enzympapper for indikering av kolinesterashemmare der enzymet immobiliserats av en anjonbytare, forfarande for dess framstellning samt anvendning av pappret med ett substrat
Huang et al. d-Serine Dehydratase from Escherichia coli: IV. COMPARATIVE SEQUENCES OF PYRIDOXYLPEPTIDES DERIVED FROM THE ACTIVE SITE AND FROM AN INHIBITORY SITE OF THE ENZYME
Nolan et al. The dissociation of rabbit reticulocyte ribosomes with EDTA and the location of messenger ribonucleic acid

Legal Events

Date Code Title Description
AS Assignment

Owner name: PIERCE CHEMICAL COMPANY, ILLINOIS

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:MALLIA, KRISHNA;REEL/FRAME:007539/0938

Effective date: 19940408

STCF Information on status: patent grant

Free format text: PATENTED CASE

FPAY Fee payment

Year of fee payment: 4

FPAY Fee payment

Year of fee payment: 8

AS Assignment

Owner name: PIERCE BIOTECHNOLOGY, INC., ILLINOIS

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:PIERCE CHEMICAL COMPANY;REEL/FRAME:014845/0752

Effective date: 20020320

AS Assignment

Owner name: PIERCE BIOTECHNOLOGY, INC., ILLINOIS

Free format text: CHANGE OF NAME;ASSIGNOR:PIERCE CHEMICAL COMPANY;REEL/FRAME:014934/0359

Effective date: 20020320

AS Assignment

Owner name: DEUTSCHE BANK AG, NEW YORK BRANCH, NEW YORK

Free format text: SECURITY INTEREST;ASSIGNOR:PIERCE BIOTECHNOLOGY, INC.;REEL/FRAME:015008/0214

Effective date: 20040123

AS Assignment

Owner name: PIERCE BIOTECHNOLOGY, INC., ILLINOIS

Free format text: RELEASE OF SECURITY INTEREST;ASSIGNOR:DEUTSCHE BANK AG NEW YORK BRANCH;REEL/FRAME:015756/0294

Effective date: 20040802

Owner name: HYCLONE LABORATORIES, INC., UTAH

Free format text: RELEASE OF SECURITY INTEREST;ASSIGNOR:DEUTSCHE BANK AG NEW YORK BRANCH;REEL/FRAME:015756/0294

Effective date: 20040802

FPAY Fee payment

Year of fee payment: 12